Gene Expression

What is your primer-pair disqualification sequence?

by Lale

Before expression analysis, in what order do you test a new qPCR primer pair, and which result makes you discard it rather than optimize further? I would compare in silico specificity, no-template and no-RT controls, melt behavior, product size or identity, amplification efficiency across a dilution series, and performance in the intended sample matrix. Please include the concrete thresholds you use, especially for efficiency and nonspecific amplification, before selecting reference targets or calculating normalized expression.

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Miro K.

My sequence is in silico specificity, NTC and no-RT controls, then melt curve plus gel or product sequencing, followed by a dilution series in the intended matrix. I discard the pair if it produces a reproducible off-target product, a primer-dimer signal that overlaps the target range, or genomic amplification that cannot be separated by redesign. For efficiency, I use 90% to 110% as a working acceptance range, but also inspect linearity, replicate scatter, and inhibition rather than accepting the slope alone. MIQE requires empirical specificity, controls, and reported efficiency, but does not supply one universal disqualification threshold. Which of efficiency, melt behavior, and negative controls is already known?

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Lale

None of those results is known from the discussion yet. I would compare the same primer pair across a dilution series in the intended matrix and a matched clean template series. If dilution of the matrix restores Cq linearity and moves apparent efficiency toward the working range, inhibition is the stronger explanation. If the abnormal slope persists with clean template while specificity remains acceptable, primer kinetics becomes the stronger reason to redesign.

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Miro K.

That resolves what is currently known: none of the three checks has been run. Check the NTC, no-RT control, and melt behavior first, since paired matrix and clean-template slopes are interpretable only if both series measure the intended product.

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Lale

My earlier comment said the results weren't known from this discussion; it didn't establish that the checks hadn't been run. The NTC, no-RT and melt results remain unreported here. Your proposed order addresses what to examine before comparing slopes, but we still can't say whether those checks need doing or whether existing results need to be shared.

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